Concerted Structural Changes in the Peptidase and the Propeller Domains of Prolyl Oligopeptidase are Required for Substrate Binding

Szeltner, Zoltán; Rea, Dean; Juhász, Tünde; Renner, Veronika; Fülöp, Vilmos; Polgár, László

doi:10.1016/j.jmb.2004.05.011

Cited by 55 publications

(64 citation statements)

References 53 publications

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“…Porcine muscle POP is a serine oligopeptidase with an unusual ␤-propeller domain. This domain is thought to limit access of larger polypeptides to the active site, and this is consistent with porcine muscle POP acting on oligopeptide substrates, Indeed, an engineered disulfide "latch" on the ␤-propeller lid abolishes even oligopeptidase activity (52). This is consistent with the notion that PCY1 also acts on oligopeptides as shown in this paper, although further work is required to determine the limits of its substrate size.…”

Section: Discussionsupporting

confidence: 86%

The Two-step Biosynthesis of Cyclic Peptides from Linear Precursors in a Member of the Plant Family Caryophyllaceae Involves Cyclization by a Serine Protease-like Enzyme

Barber

Pujara

Reed

et al. 2013

Journal of Biological Chemistry

140

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Background: In the Caryophyllaceae, cyclic peptides (CP) are biosynthesized from linear precursors via an unknown pathway. Results: Two protease-like enzymes are involved in precursor processing. Conclusion: A serine protease-like enzyme was recruited for the cyclization step in CP biosynthesis. Significance: This represents a very significant advance in our understanding of the mode and evolution of CP biosynthesis in plants.

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Section: Discussionsupporting

confidence: 86%

The Two-step Biosynthesis of Cyclic Peptides from Linear Precursors in a Member of the Plant Family Caryophyllaceae Involves Cyclization by a Serine Protease-like Enzyme

Barber

Pujara

Reed

et al. 2013

Journal of Biological Chemistry

140

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“…In contrast, only an 11.5-fold increase in K m and a slightly increased k cat was found for the T202C/T590C variant. Binding of the octapeptide to the almost inactive loop A -enzyme was undetectable in a titration experiment performed as in [24] (data not shown). Inhibitor binding is also drastically reduced.…”

Section: 4mentioning

confidence: 88%

“…However, the propeller domain of POP was found to be too rigid for such a regulatory function [23]. Limiting the flexibility between the propeller and catalytic domains with an engineered disulfide bridge inactivated the enzyme [24]. Flexibility of the loop structure at the domain interface was suggested to be required for efficient catalysis [24,25].…”

Section: Introductionmentioning

confidence: 99%

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The loops facing the active site of prolyl oligopeptidase are crucial components in substrate gating and specificity

Szeltner

Juhász

Szamosi

et al. 2013

Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics

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Prolyl oligopeptidase (POP) has emerged as a drug target for neurological diseases. A flexible loop structure comprising loop A (res. 189-209) and loop B (res. 577-608) at the domain interface is implicated in substrate entry to the active site. Here we determined the kinetic and structural properties of POP with mutations in loop A, loop B and in two additional flexible loops. POP lacking loop A proved to be an inefficient enzyme as did POP with a mutation in loop B (T590C). Both constructs displayed an altered substrate preference profile. Ligand binding become markedly degraded. Conversely, the T202C mutation increased the flexibility of loop A, enhancing the catalytic efficiency beyond that of the native enzyme. The T590C mutation in loop B increased the preference for shorter peptides, indicating a role in substrate gating. Loop A and the His loop housing the catalytic histidine are disordered in the H680A mutant crystal structure, implying coordinated structural dynamics of these loops. A 17-mer peptide could not inhibit variants possessing malfunctioning loop A. This substrate may bind non-productively to an exosite involving loop A or to an open enzyme form. Biophysical studies suggest that mammalian POP resides in a predominantly closed conformational state, especially at physiological conditions. The flexible loop A, loop B and His loop system at the active site is the main regulator of substrate gating and specificity and represents a new inhibitor target.

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“…In PEP, conformational change is required for catalysis and decreased propeller domain flexibility has been associated with decreased catalysis [36,41]. Moreover, an open form of PEP, from Myxococcus xanthus, has been observed in which the two domains move apart on a hinge [42].…”

Section: Discussionmentioning

confidence: 99%

Reversible Inactivation of Human Dipeptidyl Peptidases 8 and 9 by Oxidation

Park¹,

Knott²,

Nadvi³

et al. 2008

TOEIJ

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Abstract:Hydrogen peroxide (H 2 O 2 ) can act as an intracellular messenger by oxidizing sulfhydryl groups in cysteines that can be oxidized at neutral pH. The oxidizing agents H 2 O 2 and pyrroloquinoline quinone and the large thiol reagents N-ethylmaleimide and 4-(hydroxymercuri) benzoate each inhibited dipeptidyl peptidase (DP) activity in the intracellular DPIV-related proteins DP8 and DP9 at pH 7.5. In contrast, these treatments did not alter activity in DPIV and fibroblast activation protein. Peptidase inhibition was completely reversed by 2-mercaptoethanol or reduced glutathione. Alkylation of DP8 by the small thiol reagent iodoacetamide prevented inhibition by H 2 O 2, N-ethylmaleimide or pyrroloquinoline quinone. Two cysteines were reactive per peptidase monomer. We exploited these properties to highly purify DP8 by thiol affinity chromatography. Homology modelling of DP8 and DP9 was consistent with the proposal that the mechanism involves decreased protein flexibility caused by intramolecular disulfide bonding. These novel data show that DP8 and DP9 are reversibly inactivated by oxidants at neutral pH and suggest that DP8 and DP9 are H 2 O 2 sensing proteins.

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Concerted Structural Changes in the Peptidase and the Propeller Domains of Prolyl Oligopeptidase are Required for Substrate Binding

Cited by 55 publications

References 53 publications

The Two-step Biosynthesis of Cyclic Peptides from Linear Precursors in a Member of the Plant Family Caryophyllaceae Involves Cyclization by a Serine Protease-like Enzyme

The Two-step Biosynthesis of Cyclic Peptides from Linear Precursors in a Member of the Plant Family Caryophyllaceae Involves Cyclization by a Serine Protease-like Enzyme

The loops facing the active site of prolyl oligopeptidase are crucial components in substrate gating and specificity

Reversible Inactivation of Human Dipeptidyl Peptidases 8 and 9 by Oxidation

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